Cholic acid (CA) and chenodeoxycholic acid (CDCA) are biotransformed into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, by the intestinal microflora. High levels DCA and LCA in bile, blood, and feces have been associated with an increased risk of cholesterol gallstone disease and colon cancer in man. DCA may also play a role in the pathophysiology of cholestatic liver diseases. The levels and activities of CA 7alpha-dehydroxylating intestinal bacteria, intestinal transit time and pH control the concentration of DCA in the bile acid pools of humans. CA 7alpha-dehydroxylation is carried out by a tiny fraction of the intestinal microflora belonging to the genus Clostridium. This apparently straightforward reaction is in fact a biochemical pathway controlled by at least eight enzymes and transporters. In order to determine if bacteria play a role in cholesterol gallstone disease, in some patients, and to develop inhibitors (drugs) of this pathway as well as other approaches for lowering DCA in the body, we propose the following specific aims: 1) Determine the 3 dimensional (3D) structure and catalytic mechanism of bile acid 7alpha and 7beta-dehydratase from Clostridium scindens VPI 12708. The 3D structures will be determined by computer modeling and by x-ray crystallography techniques. The catalytic mechanism will be determined by site-directed mutagenesis, enzyme kinetics and substrate binding techniques; 2) Express, purify, and characterize a novel 3-oxo-delta4-steroid oxidoreductase from Clostridium scindens VPI 12708; 3) Clone, sequence, and analyze the bai operon and upstream regulatory region from Clostridium hylemonae TN271, a "low" activity CA 7alpha-dehydroxylating intestinal species; 4) Isolate, characterize, and identify (by 16S ribosomal gene sequence) CA 7alpha-dehydroxylating bacteria from cholesterol gallstone patients and controls. Determine if cholesterol gallstone patients are colonized by unique species of bile acid 7alpha-dehydroxylating bacteria.